X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG
Abstract and Figures
Uracil-DNA glycosylase (UDG), a key highly conserved DNA repair enzyme involved in uracil excision repair, was discovered
in Escherichia coli. The Bacillus subtilis bacteriophage, PBS-1 and PBS-2, which contain dUMP residues in their DNA, express a UDG inhibitor protein, Ugi which binds
to UDG very tightly to form a physiologically irreversible complex. The X-ray analysis of the E.coli UDG (EcUDG)-Ugi complex at 3.2 Å resolution, leads to the first structure elucidation of a bacterial UDG molecule. This structure
is similar to the enzymes from human and viral sources. A comparison of the available structures involving UDG permits the
delineation of the constant and the variable regions of the molecule. Structural comparison and mutational analysis also indicate
that the mode of action of the enzyme from these sources are the same. The crystal structure shows a remarkable spatial conservation
of the active site residues involved in DNA binding in spite of significant differences in the structure of the enzyme-inhibitor
complex, in comparison with those from the mammalian and viral sources. EcUDG could serve as a prototype for UDGs from pathogenic prokaryotes, and provide a framework for possible drug development
against such pathogens with emphasis on features of the molecule that differ from those in the human enzyme.
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... L'intensité de l'onde diffractée est proportionnelle au carré de son amplitude. 17 Plomb, Mercure, Platine ... 18 Carbone, azote, oxygène ... hydrogène ... ...
... This loop provides the residues that position and activate a catalytic water molecule for a nucleophilic attack on the C1′ atom of the ribose and hydrolysis of the N-glycosylic bond. 16,25 Structures have also been solved in complex with the uracil-DNA glycosylase inhibitory protein (Ugi), 18,[26][27] which is produced by Bacillus subtilis bacteriophages PBS-1 and PBS-2. 29 These phages use uracil instead of thymine in their DNA genomes, 30 which are protected from cellular UNG by the expression of Ugi. ...
Epstein-Barr Virus (EBV) is a Human γ –herpesvirus. It is the causative agent of diseases such as Infectious Mononucleosis and it is associated with several immunoproliferative syndromes. Unlike other herpesviruses such as herpes-simplex, there is no treatment against EBV. EBV genome features 86 open-reading frames which are potential targets for a structural genomics approach and for the rational design of new drugs. The first round of selected targets encode for 23 proteins. The main problem we encountered was, unexpectedly, the poor expression and solubility of the majority of the selected targets. Among the 8 soluble proteins we obtained, 5 were crystallized and 4 structures were solved. The main success factor was an individual treatment of each target rather than the use of standard protocols. The Uracil-DNA Glycosylase (UNG) is a DNA repair enzyme. It was impossible to obtain crystals of the protein alone, but crystals diffracting to 2.3 Å grew upon the formation of a complex with a phage-encoded inhibitory protein. The analysis of the structure of this complex shows that the « leucin-loop », a major domain of the active site, features a 7 residue insertion which is common to all γ –herpesvirus. Despite this difference, catalytic constants are similar to other organisms UNGs, suggesting a similar interaction with DNA. Current work and problems associated with other targets is also reported (in particular on the Alkaline Exonuclease).
... This UDG family is characterized by motif A (GQDPY) responsible for catalysis and motif B (HPSPLS) implicated in the stabilization of the enzyme substrate complex (11)(12)(13)(14). Interestingly, phage PBS-1 or 2 that infects Bacillus subtilis encodes a protein inhibitor, Ugi that forms a physiologically irreversible non-covalent complex in 1:1 stoichiometry specifically with family 1 UDGs to allow occurrence of uracil in their genomes (15,16). ...
U:
Repair of uracils in DNA is initiated by racil NA lycosylases (UDGs). Family 1 UDGs (Ung) are the most efficient and ubiquitous proteins having an exquisite specificity for uracils in DNA. Ung are characterized by motifs A (GQDPY) and B (HPSPLS) sequences. We report a novel dimeric UDG, Blr0248 ( Bdi Ung) from Bradyrhizobium diazoefficiens . Although Bdi Ung contains the motif A (GQDPA), it has low sequence identity to known UDGs. Bdi Ung prefers single stranded DNA and excises uracil, 5-hydroxymethyl-uracil or xanthine from it. Bdi Ung is impervious to inhibition by AP DNA, and Ugi protein that specifically inhibits family 1 UDGs. Crystal structure of Bdi Ung shows similarity with the family 4 UDGs in its overall fold but with family 1 UDGs in key active site residues. However, instead of a classical motif B, Bdi Ung has a uniquely extended protrusion explaining the lack of Ugi inhibition. Structural and mutational analyses of Bdi Ung have revealed the basis for the accommodation of diverse substrates into its substrate binding pocket. Phylogenetically, Bdi Ung belongs to a new UDG family. Bradyrhizobium diazoefficiens presents a unique scenario where the presence of at least four families of UDGs may compensate for the absence of an efficient family 1 homologue.
... A common characteristic of UNGs is their inhibition by Ugi (Zharkov et al., 2010), and a large number of crystal structures have been determined of UNG in complex with Ugi (Mol, Arvai, Sanderson et al., 1995;Putnam et al., 1999;Ravishankar et al., 1998;Saikrishnan et al., 2002;Gé oui et al., 2007;Kaushal et al., 2008;Bañ os-Sanz et al., 2013;Wang et al., 2014;Assefa et al., 2014). The complex structures have revealed that the UNG-Ugi interaction closely resembles the interaction of UNG with DNA and has provided an alternative strategy for understanding the nature of UNG-DNA interaction. ...
Uracil-DNA it N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium it Deinococcus radiodurans (it DrUNG) in complex with DNA is reported at a resolution of 1.35Å. Prior to the crystallization experiments, the affinity between it DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme--DNA interaction. The complex revealed that the interaction between it DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug it et al. (1996). it Nature (London), bf 384, 87--92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in it DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of it DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues in the N-terminus of a symmetry-related molecule and the complementary DNA strand facing away from the active site were also observed which seem to stabilize the enzyme--DNA complex. However, the significance of this observation remains to be investigated. The results provide new insights into the current knowledge about DNA damage recognition and repair by uracil-DNA glycosylases.
... Ung proteins, which are amongst the most efficient enzymes are characterized by motif A sequence, GQDPY involved in substrate catalysis, and motif B sequence, HPSPLS involved in stabilizing the enzyme substrate complex (3,(18)(19)(20). Family 1 UDGs (Ung) are specifically inhibited by B. subtilis phage PBS-1 or 2 encoded proteinaceous inhibitor called Ugi (uracil DNA glycosylase inhibitor) by forming a physiologically irreversible non-covalent complex in 1:1 stoichiometry (21,22). Family 2 UDGs (Mug/TDG) are mismatch specific DNA glycosylases which excise thymine from T:G pair and possess GINPG and MPSSAR as motifs A and B sequences, respectively. ...
Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
... Although inhibition by these small molecules has been well studied biochemically, the modes of their binding and interactions with the enzyme have not been extensively explored. Structures of free UNG/Ung from many sources and their complexes with Ugi have been reported (Mol, Arvai, Sanderson et al., 1995;Ravishankar et al., 1998;Xiao et al., 1999;Putnam et al., 1999;Saikrishnan et al., 2002;Leiros et al., 2003Leiros et al., , 2005Moe et al., 2004;Gé oui et al., 2007;Raeder et al., 2010;Assefa et al., 2012Assefa et al., , 2014. The structure of the complex with oligonucleotides, however, is known only for the human (Slupphaug et al., 1996;Parikh et al., 1998Parikh et al., , 2000Bianchet et al., 2003), Escherichia coli (Werner et al., 2000) and Herpes simplex virus 1 (HSV1; enzymes. ...
17 independent crystal structures of family I uracil-DNA glycosylase from
Mycobacterium tuberculosis
(
Mt
Ung) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5′ position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.
... The study of DNA repair systems, of which RecA is a crucial component, has been of great interest in recent years and is likely to be particularly important in mycobacteria (46). Although structures of some of the proteins involved in one or other pathways of DNA repair have been characterised (47,48), such information in mycobacteria is still very scant. Also, in bacteria the biological role of homologous genetic recombination is predominantly DNA repair under normal growth conditions. ...
Sequencing of the complete genome of Mycobacterium tuberculosis, combined with the rapidly increasing need to improve tuberculosis management through better drugs and vaccines, has initiated
extensive research on several key proteins from the pathogen. RecA, a ubiquitous multifunctional protein, is a key component
of the processes of homologous genetic recombination and DNA repair. Structural knowledge of MtRecA is imperative for a full
understanding of both these activities and any ensuing application. The crystal structure of MtRecA, presented here, has six
molecules in the unit cell forming a 61 helical filament with a deep groove capable of binding DNA. The observed weakening in the higher order aggregation of filaments
into bundles may have implications for recombination in mycobacteria. The structure of the complex reveals the atomic interactions
of ADP–AlF4, an ATP analogue, with the P-loop-containing binding pocket. The structures explain reduced levels of interactions of MtRecA
with ATP, despite sharing the same fold, topology and high sequence similarity with EcRecA. The formation of a helical filament
with a deep groove appears to be an inherent property of MtRecA. The histidine in loop L1 appears to be positioned appropriately
for DNA interaction.
... The crystal structure of UNG in complex with Ugi has been determined for UNGs from human (Mol, Arvai, Sanderson et al., 1995), herpes simplex virus 1 (Savva & Pearl, 1995), E. coli (Saikrishnan et al., 2002;Ravishankar et al., 1998;Putnam et al., 1999), Epstein-Barr virus (Gé oui et al., 2007) and Mycobacterium tuberculosis (Kaushal et al., 2008). These complex structures revealed that the UNG-Ugi interaction closely resembles the interaction of UNG with DNA (Mol, Arvai, Figure 1 (a) The cUNG-Ugi complex (cUNG in blue, Ugi in green) superimposed on the hUNG-Ugi complex (PDB entry 1emh; grey) with some loops labelled. ...
Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme–substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG–Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG–Ugi complex with previously determined structures of UNG–Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (K
b) which is one order of magnitude larger than that for hUNG–Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein–Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.
An autonomous nonenzymatic DNA machine is successfully engineered based on two-layered cascaded hybridization chain reaction (C-HCR) circuit, in which the tandem triggers output of upstream HCR-1 unit activate downstream HCR-2...
Residue types at the interface of protein-protein complexes are known to be reasonably well conserved. However we show, using a dataset of known 3-D structures of homologous transient protein-protein complexes, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homologue. Such differences in homologous complexes are manifested by substitution of the residues which are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient protein-protein complexes are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modelled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modelling of protein-protein complex structures. This article is protected by copyright. All rights reserved.
© 2015 The Protein Society.
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DNA glycosylases are ubiquitous in nature and essentially hydrolyze nucleoside base–glycosidic bonds. The reaction results in the production of an apurinic/apyrimidinic (AP) site in DNA and a free nucleoside base. These enzymes also participate in DNA repair processes by catalyzing the removal of unconventional or damaged bases from DNA. Six different DNA glycosylase activities are known and are divided into two main classes: (1) class I enzymes are specific for uracil, hypoxanthine, 3-methyladenine, and formamidopyrimidine residues in DNA and (2) class II enzymes appear to have an associated AP endonuclease activity that is an integral part of the enzyme. This chapter deals with the class I glycosylases. A common class I glycosylase enzyme, uracil-DNA glycosylase, has been identified in every tissue that has been examined with the exception of Drosophila. Human cells, and probably other eukaryotic cells, have both a nuclear and a mitochondrial uracil glycosylase. The DNA glycosylase reaction is mechanistically similar to other glycohydrolases and nucleosidases, such as lysozyme and β-galactosidase. All of the DNA glycosylases are potentially useful for detecting specific lesions in DNA by either the direct measurement of released bases or by the indirect but more sensitive method of introducing alkali-sensitive AP sites into DNA. Uracil glycosylase has been used to detect uracil residues formed by treating DNA with heat, NaHSO3, or nitrous acid.
Bond-length and bond-angle parameters are derived from a statistical survey of X-ray structures of small compounds from the Cambridge Structural Database. The side chains of the common amino acids and the polypeptide backbone were represented by appropriate chemical fragments taken from the Database. Average bond lengths and bond angles are determined from the resulting samples and the sample standard deviations provide information regarding the expected variability of the average values which can be parametrized as force constants. These parameters are ideally suited for the refinement of protein structures determined by X-ray crystallography since they are derived from X-ray structures, are accurate to within the deviations from target values suggested for X-ray structure refinement and use force constants which directly reflect the variability or uncertainty of the average values. Tests of refinement of the structures of BPTI and phycocyanin demonstrate the integrity of the parameters and comparisons of equivalent refinements with XPLOR parameters show improvement in R factors and geometry statistics.
A model building and refinement system is described for use with a Vector General 3400 display. The system allows the user to build models using guide atoms and angles to arrive at the final conformation. It has been used to assist in difference Fourier map interpretation at medium and high resolution, and to build a protein molecule into a multiple isomorphous replacement phased electron density map.
A single-stage computer procedure to calculate an electron density map suitable to detect errors in a tentative macromolecular model has been developed. In this procedure, an atom of the tentative model does not contribute to the phases used to calculate electron density values at or near its current position, that is within the region containing it and a neighborhood surrounding that region. In this way, the phases used to calculate electron density values within a region are not biased by the model atoms contained within that region or its neighborhood. The number of atoms which are omitted for a given region is maintained at a small fraction of the total structure so that the phases used to calculate electron density values may still be a good approximation to the phases of the complete structure. The procedure was used to improve the model of the Fab portion of the mouse galactan-binding immunoglobulin J539 (IgA2, κ), which contains 431 residues.
For a successful analysis of the relation between amino acid sequence and protein structure, an unambiguous and physically meaningful definition of secondary structure is essential. We have developed a set of simple and physically motivated criteria for secondary structure, programmed as a pattern-recognition process of hydrogen-bonded and geometrical features extracted from x-ray coordinates. Cooperative secondary structure is recognized as repeats of the elementary hydrogen-bonding patterns “turn” and “bridge.” Repeating turns are “helices,” repeating bridges are “ladders,” connected ladders are “sheets.” Geometric structure is defined in terms of the concepts torsion and curvature of differential geometry. Local chain “chirality” is the torsional handedness of four consecutive Cα positions and is positive for right-handed helices and negative for ideal twisted β-sheets. Curved pieces are defined as “bends.” Solvent “exposure” is given as the number of water molecules in possible contact with a residue. The end result is a compilation of the primary structure, including SS bonds, secondary structure, and solvent exposure of 62 different globular proteins. The presentation is in linear form: strip graphs for an overall view and strip tables for the details of each of 10.925 residues. The dictionary is also available in computer-readable form for protein structure prediction work.
Escherichia coli cells containing elevated levels of the DNA repair enzyme uracil-DNA glycosylase (the ung gene product) have been constructed by in vitro recombination methods. First, λnadB transducing phages were isolated from two E. coli DNA libraries by selection of nicotinate-independent lysogens. λnadB phage from one of the libraries were also ung+ and carried the ung-nadB genes on an 8.3 kb HindIII restriction fragment. The ung and nadB genes were subcloned into plasmids and a restriction map of the ung region of the E. coli chromosome was constructed. The uracil glycosylase gene was localized to a 1.4-kb restriction fragment by subcloning the gene into pBR322. Uracil glycosylase was overproduced (relative to the specific activity of wild type cells) by about two-fold in λung lysogens and by 15- to 20-fold in cells containing pBR322ung derivatives. When the ung gene and its promoter were placed downstream from the bacteriophage promoter in the plasmid pKC30, uracil glycosylase production was heat-induced to more than 100-fold above the levels of a wild-type cell. By relating the insertion orientation of the λung gene in the plasmid pKC30 to its orientation in λung-nadB transducing phages, the transcription direction of the ung gene on the E. coli linkage map was found to be clockwise.
Many organisms expand the information content of their genome through enzymatic methylation of cytosine residues. Here we report the 2.8 A crystal structure of a bacterial DNA (cytosine-5)-methyltransferase (DCMtase), M. HaeIII, bound covalently to DNA. In this complex, the substrate cytosine is extruded from the DNA helix and inserted into the active site of the enzyme, as has been observed for another DCMtase, M. HhaI. The DNA is bound in a cleft between the two domains of the protein and is distorted from the characteristic B-form conformation at its recognition sequence. A comparison of structures shows a variation in the mode of DNA recognition: M. HaeIII differs from M. HhaI in that the remaining bases in its recognition sequence undergo an extensive rearrangement in their pairing. In this process, the bases are unstacked, and a gap 8 A long opens in the DNA.